Aim 1: The mechanism of gelation. Mechanistically, two distinct models can describe the events leading to gelation that differ in their early steps. Mechanism 1 asserts that monomeric peptides first fold into discreet amphiphilic beta-hairpins that then associate facially and laterally to form fibrils. We initially favored mechanism 1 based on early data and literature describing hairpin folding. However, mechanistic studies of amyloid forming peptides and intrinsically disordered proteins suggest that the early steps may involve the formation of micelle-like oligomers (mechanism 2). Here, the rapid association of unfolded peptides is driven by hydrophobic collapse to form oligomers, which may act to increase the local concentration of peptide and facilitate their ordering to initiate folding and assembly into beta-rich fibrils. Either mechanism could lead to the evolution of clusters of well-defined fibrils, which we directly observe by cryo-TEM at later times. Prior work showed that individual clusters contain dangling fibril ends that grow and interpenetrate neighboring clusters as the network evolves. The exact time at which the clustered fibril network percolates the entire sample volume and the solution becomes a gel is fast (1min at 1 wt% peptide) and concentration dependent. After the gel point, the network continues to grow, filling the voids, to further rigidify the gel. Cryo-TEM suggests that the final network contains fibrils that entangle and form branch-points, both are physical crosslinks that help define the gel's mechanical properties. The mesh size of the network can be varied (20-50 nm) by adjusting the peptide concentration or the rate of self-assembly. In general, faster rates of assembly lead to more crosslinks, smaller mesh sizes, and stiffer gels. With respect to drug delivery, this range of mesh sizes is similar to the diameters of many therapeutic proteins and thus, influences their release behavior from the gel. Aim 2: Molecular design of peptide hydrogels. We continuously design new peptides to refine our understanding of how sequence composition affects material formation and properties. Previously, we found that strand number and strand registry influence local fibril morphology, and that two-stranded symmetrical beta-hairpins reproducibly assemble into fibrils having consistent morphology that form mechanically well-defined gels best suited for delivery applications. We found that changes in residue composition on the hairpin's hydrophilic face that reduce charge density promotes folding, assembly, and the formation of stiffer gels. Thus, minimally charged peptides form gels at lower values of solution pH, ionic strength and temperature. Further, the hairpin's hydrophilic face can accommodate nearly any natural or non-natural residue without affecting fibril formation and gelation. Aim 3: Molecular-level structure of peptide fibrils and their networks. Previous work gave us an understanding of the local fibril structure largely based on models derived from TEM, AFM, and SANS data, but no detail with respect to the exact molecular arrangement of peptides in the assembly. Further, we have little data directly reporting on the network-level structure of the gel; how do the fibrils associate to form a network? Do they simply entangle, do they form branches (as we have proposed), and do remnants of oligomers formed early in the gelation mechanism persist in the network? Aim 4: Study the physical interactions between fibrillar network and encapsulated therapy. Small molecules, proteins, RNA, DNA and cells can be directly encapsulated in the gel network by adding a solution of unfolded peptide in water to a solution of therapy in triggering buffer. Physical interactions between the therapy and the fibril network determine how each therapy type partitions within the gel during its encapsulation, and influences the rate at which it is released. Our work suggests that the rules governing these processes differ according to therapy type. Aim 5: Develop peptide materials towards clinical applications. Our basic science lab looks towards the clinic for inspiration, leading to several applied projects, including mesothelioma, tissue transplantation and immune modulation.